Dataset Information

Chromatin immunoprecipitation of ectopic FLAG-tagged TCF7L1 followed by Mass Spectrome-try in H9-TCF7L1 Human Embryonic Stem Cells

ABSTRACT: TCF7L1 is a member of the T cell factor/Lymphoid enhancer factor (TCF/LEF) family of tran-scription factors that are part of the WNT/beta-CATENIN signaling pathway. TCF7L1 modulates transcription by interacting with other regulators on chromatin. TCF7L1 has been shown to be one of the key factors in maintaining pluripotency in human embryonic stem cells (ESCs). We previously demonstrated that the absence of TCF71 causes H9 hESCs to differentiate sponta-neously (Sierra et al., 2018 Development 145(4).pii:dev161075). Mechanistically, how TCF7L1 inhibits differentiation and keeps cells in the pluripotent state is not clear. Identifying transcrip-tional regulators on chromatin is a critical step to elucidating one of several molecular mecha-nisms controlled by TCF7L1. We previously developed a FLAG tagged TCF7L1 transgene that is controlled by a doxycycline-inducible TET-ON system and generated the H9-TCF7L1 line (Sier-ra et al., 2018 Development 145(4).pii:dev161075). Here we used the H9-TCF7L1 line and em-ployed the method called rapid immunoprecipitation (IP) mass spectrometry of endogenous pro-tein (RIME) (Mohammed et al., 2016 Nat Protoc 11(2):316-26) to identify TCF7L1-associated proteins on chromatin. Coupling of these two methods (the FLAG tagged TETON inducible sys-tem and RIME) allowed us to control the level of TCF7L1 expression in hESCs, immunopreci-pate TCF7L1 using an anti-FLAG antibody and capture TCF7L1-bound associated complexes on chromatin. Our MS analysis identified some known proteins that have been shown to associate with the WNT/beta-CATENIN/TCF/LEF pathway, as well as novel complexes that have not been linked with TCF7L1. Gene Ontology analysis suggest these proteins function in chromatin modi-fication, splicing, and RNA processing. Our data could create new ideas for in-depth studies of TCF7L1 controlling pluripotency in human ESCs and for understanding how TCF7L1 may act in other cell types.

INSTRUMENT(S): Orbitrap Fusion, nanoACQUITY UPLC

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Linh Vuong

LAB HEAD: Peter J. Donovan

PROVIDER: PXD047582 | Pride | 2024-06-16

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Linh_TCF3_6samples_Dec2016_MudPIT5_PD2.2.msf	Msf
	Linh_TCF3_6samples_Dec2016_MudPIT5_PD2.2.msfView	Msf
	Linh_TCF3_6samples_Dec2016_MudPIT5_PD2.2_Mod.msf	Msf
	Linh_TCF3_6samples_Dec2016_MudPIT5_PD2.2_Mod.msfView	Msf
	Linh_TCF3_6samples_Dec2016_MudPIT5_PD2_2.mzTab	Mztab

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Publications

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Vuong Linh M LM Pan Songqin S Sierra Robert A RA Waterman Marian L ML Gershon Paul D PD Donovan Peter J PJ

Proteomics 20240417 20

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output ...[more]

PMID: 38629187

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